Toll-like receptor 9: trafficking and signaling

摘要 含CpG基序的DNA（CpG DNA）具有治疗癌症、传染病和过敏性疾病的巨大潜力 疾病尽管有这种潜力，对我们自己的核酸，包括DNA的反应，触发自身免疫 系统性红斑狼疮等疾病。特异性受体TLR 9的定位和运输， 可能在自身/外源DNA辨别中起关键作用。揭示TLR 9的分子机制 贩运是我们操纵TLR 9贩运以实现长期目标的第一步。 CpG DNA反应的调节。我们已经证明TLR 9定位于细胞内，主要在 内质网（ER），在CpG DNA刺激之前。TLR 9从ER运输到内体， 在溶酶体中与内吞的CpG DNA共定位。这些数据提出了以下基本问题： 什么调控TLR 9进入CpG DNA，以及进入如何影响对自身和微生物DNA的反应？ 使用新的TLR 9运输测定，我们已经积累了TLR 9组成型退出ER的证据， CpG DNA诱导了二次TLR 9运输事件，这两个事件都是通过传统的细胞内 贩运途径。因此，我们假设TLR 9组成性地以高度依赖性的方式运输。 调节方式通过细胞分泌途径从高尔基复合体， 内溶酶体和高尔基体转运是TLR 9应答CpG DNA的先决条件。我们 我相信TLR 9的运输可能与其他TLRs和自身免疫性B协同作用 细胞受体，并通过TLR 9胞质尾区的翻译后修饰进行调节。 这一假设将在三个具体目标中进行检验。首先，转染的和内源性的TLR 9运输 通过高尔基复合体将使用多种体外方法，包括一种新的蛋白酶， 裂解测定其次，我们将研究TLR 9-自身免疫B细胞受体协同作用的机制。 第三，我们将确定TLR 9的翻译后修饰在调节细胞内信号转导中的作用。 运输和TLR-自身免疫B细胞受体协同作用。通过了解TLR 9的机制， 我们可以揭示如何调节对外来DNA的反应和缺乏对自身DNA的反应， 办妥了一批这些知识将为进一步开发CpG DNA作为佐剂提供基础， 治疗以及开发机制，中断自身免疫病理循环。

ABSTRACT DNA containing CpG motifs (CpG DNA) has incredible potential to treat cancer, infectious and allergic diseases. Despite this potential, response to our own nucleic acids, including DNA, triggers autoimmune diseases such as systemic lupus erythematosus. Localization and trafficking of the specific receptor, TLR9, may play a key role in self/foreign DNA discrimination. Uncovering the molecular mechanisms of TLR9 trafficking is the first step towards our long term goal of manipulating TLR9 trafficking to achieve modulation of CpG DNA response. We have shown that TLR9 is localized intracellularly, predominantly in the endoplasmic reticulum (ER), prior to CpG DNA stimulation. TLR9 traffics from the ER to endosomes and lysosomes where it co-localizes with endocytosed CpG DNA. These data raise the fundamental questions of what regulates TLR9 access to CpG DNA and how does access affect response to self and microbial DNA? Using new TLR9 trafficking assays, we have accumulated evidence that TLR9 constitutively exits the ER, that CpG DNA induces a secondary TLR9 trafficking event, and that both events occur through traditional cell trafficking pathways. Therefore, we hypothesize that TLR9 constitutively traffics in a highly regulated fashion through the cell secretory pathway from the Golgi complex to endolysosomes and that Golgi transit is a prerequisite for TLR9 response to CpG DNA. We believe that TLR9 trafficking may account for synergy with other TLRs and the autoimmune B cell receptor, and is regulated by post-translational modification of the TLR9 cytoplasmic tail. This hypothesis will be tested in three Specific Aims. First, transfected and endogenous TLR9 trafficking through the Golgi complex will be examined using multiple in vitro approaches including a novel protease cleavage assay. Second, we will examine the mechanism of TLR9-autoimmune B cell receptor synergism. Third, we will determine the role of TLR9 post-translational modification in regulating intracellular trafficking and in TLR-autoimmune B cell receptor synergy. By understanding the mechanism of TLR9 trafficking we can uncover how regulation of response to foreign DNA and lack of response to self-DNA is achieved. This knowledge will provide the basis for the further development of CpG DNA as an adjuvant and therapeutic as well as develop mechanisms to interrupt the cycle of autoimmune pathology.